Note: Descriptions are shown in the official language in which they were submitted.
CA 02393223 2002-03-28
Multiplex PCR for detecting EHEC infections
The present invention originates from the diagnostic area of the detection of
hemorrhagic diarrheal diseases.
Enterohemorrhagic E. coli pathogens (EHEC) are dangerous pathogens of
diarrheal diseases which may be transmitted both by foodstuffs and by smear
infections. Enterohemorrhagic E. coli organisms are able to form highly potent
cytotoxins. These are proteins which have great similarity with the Shiga
toxin of
Shigella dysenteriae type 1 and are therefore called Shiga toxin 1 and Shiga
toxin 2.
The genes coding therefor for the respective subunits A and B are referred to
as stxA1
GenBank number M19473) and stxB1 GenBank number M19473) and stxA2 (GenBank
number X07865) and stxB2 GenBank number X07865). Pathogenic E. coli strains
may
contain either one of the two Shiga toxin genes or else both. In addition, the
pathogens
may have further associated virulence factors such as EHEC intimin, EHEC
hemolysin,
EHEC catalase, EHEC serine protease and EHEC enterotoxin.
The diagnostic detection of EHEC is, because of the transmission routes and of
the low infectious dose of about 10z-103 organisms, important not only for
those with
acute disease but also in order to identify possible excretors or find other
sources of
infection. In the state of the art,EHEC infections are detected by
microbiological means
using sorbitol McConkey selective agar plates or by means of toxin ELISAs. In
addition,
PCR detection methods exist and can be used to detect Shiga toxin gene
sequences
(for example Chen et al., Applied and Environmental Microbiology Vol. 64 No.
11, pp.
4210-4116, 1998; Gannon et al., Applied Environmental Microbiology, Vol. 58
No. 12,
pp. 3809-3815, 1992; Pierard et al., Journal of Clinical Microbiology Vol. 36,
No. 11, pp.
CA 02393223 2002-03-28
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3317-3322,). However, the only methods currently used in routine diagnosis are
those
which amplify the sequence coding for the B subunit.
Sequence analysis of the Shiga toxin genes from various isolates has shown
that not only are the primary sequences of Shiga toxin 1 and Shiga toxin 2
different
from one another, but that, in addition, different EHEC isolates in which
Shiga toxin 2
can be detected immunologically also display different alleles in relation to
the
sequence of the corresponding gene. Thus, the only primer sequences suitable
for
diagnostic detection of Shiga toxin 2 are those which are directed against
highly
conserved regions within the Shiga toxin genes and thus are able to detect the
sequences of all the alleles.
In addition to the classical diarrhea) diseases caused by known human-
pathogenic EHEC pathogens, there exist other diarrhea) diseases with a similar
clinical
picture. It has likewise been possible to diagnose enterohemorrhagic pathogens
as the
cause of these diseases by tests with Vero cell cultures (Pierard et al.,
Lancet 338,
p. 762, 1991; Thomas et al., Eur. J. Clin. Microbiol. Infect. Dis. 13, pp.
1074-1076,
1994). However, these infections were not detectable by prior art molecular
methods for
detecting human-pathogenic EHEC infections (Pierard et al., J. Clin.
Microbiol. 36,
pp. 3317-3322, 1998). Since corresponding immunological tests have only
limited
specificity, elaborate microbiological enrichment methods and subsequent
molecular
sequence analyses were carried out. It was thus possible to identify as
enterohemorrhagic E. coli strains such pathogens which were previously known
only as
Shiga toxin-producing swine-pathogenic organisms (Pierard et al., Lancet 338,
p. 762,
1991; Thomas et al., Eur. J. Clin. Microbiol. Infect. Dis. 13, pp. 1074-1076,
1994). The
CA 02393223 2002-03-28
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sequences of the Shiga toxin genes of these swine-pathogenic isolates differ
distinctly
from those of human-pathogenic strains and are referred to as stx28 (Weinstein
et al., J.
Bacteriol. 170, pp. 4223-4230, 1988; Franke et al., Journal of Clinical
Microbiology
Vol. 33 No. 12, pp. 3174-3178, 1995).
Thus, to date there is no simple immunological or molecular biological method
which can be used to detect the responsible pathogens of all enterohemorrhagic
diarrheal diseases in humans.
It is therefore an object of the present invention to provide a method with
which
both human-pathogenic and swine-pathogenic EHEC pathogens can be identified by
a
single detection reaction.
This object is achieved by a nucleic acid amplification reaction for the
detection
of clinically relevant EHEC infections, with which it is possible
simultaneously to identify
stxA1 and stxA2 sequences which are derived both from human-pathogenic and
from
swine-pathogenic pathogens.
The invention thus relates to a method with which, in a multiplex
amplification
reaction for the detection of clinically relevant EHEC infections, both stxA1
and stxA2
sequences are amplified, and which is characterized in particular by
amplification both
of human-pathogenic stxA2 isoforms and of swine-pathogenic stxA2e isoforms. In
this
connection, the term "multiplex amplification reaction" refers to PCR methods
in which
at least two different primer pairs are used, one primer pair being used to
amplify stx1
sequences and a second primer pair being used to amplify stx2 sequences. The
term
"swine pathogen" is used within the scope of this application for pathogens
which have
a Shiga toxin gene stx2e (Weinstein et al., J. Bacteriol. 170, pp. 4223-4230,
1988),
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GenBank number: M21534) and primarily cause edema disease in swine, but may
also
lead to diarrheal diseases and extraint~stinal disease manifestations in
humans.
Primers which have proved particularly suitable for carrying out the method of
the invention have a length of 17-25 nucleotides, whose sequences is either
identical to
a sequence as shown in SEQ ID No. 1-4, whose sequences represent continuous
part-
sequences of one of the sequences as shown in SEQ ID No. 1-4, or in which a
sequence as shown in SEQ ID No. 1-4 forms a continuous part-sequence of the
primer.
The use of one, preferably more than one, particularly preferably all, of the
primers of
the invention for detecting EHEC infections is likewise an aspect of the
invention.
Thus, multiplex amplification reactions in which either one, more than one or
all
of the primers of the invention are used have proved advantageous for
detection of
clinically relevant EHEC infections.
The primers ordinarily used are chemically synthesized deoxyribonucleotides.
However, it is also possible in principle to employ other nucleic acid
molecules or their
derivatives such as, for example, PNA (peptide nucleic acids). In addition,
primers of
the invention can also be conjugated to detectable or immobilizable molecules.
In a specific embodiment of the method of the invention, the product of the
amplification is, in order to increase the' sensitivity of the assay,
additionally detected by
hybridization. The hybridization probes suitable for this purpose preferably
have a
length of 25-35 nucleotides. Chemically synthesized deoxyribonucleotides are
likewise
ordinarily used, but may optionally be replaced by other nucleic acid
molecules or their
derivatives such as, for example, PNA (peptide nucleic acids). By definition,
these
CA 02393223 2002-03-28
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probes are conjugated to a detectable label. This may be, for example, a
fluorescent
dye, an enzyme, a radioactive atom or a group detectable by mass spectrometry.
The invention therefore likewise 'relates to hybridization probes having a
sequence or part-sequence as shown in SEQ ID No. 5-8. However, it has proved
to be
advantageous if a hybridization probe has a sequence which is identical or
complementary to a region of the stxA1 gene or of the stxa2 gene which
corresponds to
the enzymatically active site of the polypeptide chain encoded by these genes.
In the
sequence of stxA2, this active site is located at nucleotide position 803-805
(Jackson et
al, J. Bacteriol. 172, pp. 3346-3350, 1990). Hybridization probes having a
sequence or
part-sequence as shown in SEQ ID No. 8 are thus particularly preferred.
In a further particular embodiment, the multiplex amplification products
obtained
according to the invention are detected by means of fluorescence detection.
Given the
choice of a suitable fluorescent agent, it can be added even to the PCR
mixture without
impairing the amplification efficiency. This can take place, for example, by
carrying out
the PCR reaction in the presence of a fluorescent compound which, on binding
to
double-stranded DNA molecules and on excitation with light of a suitable
wavelength,
emits fluorescent signals (WO 97/46707):
The invention thus also relates to a method in which the multiplex
amplification
products are detected with the aid of a compound which fluoresces on binding
to
double-stranded DNA. For example, the fluorescent dye SybrGreen can be
employed
for a method of this type (WO 97/46714).
The present invention further relates to a method in which the stx sequences
are
detected with the aid of one or more fluorescence-labeled hybridization
probes. Various
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embodiments are possible in this case, such as, for example, the use of
molecular
beacons (WO 95/13399, US patent No. 5 118 801 ) or so-called TaqMan probes (WO
96/34983).
Also suitable for the quantitative detection of nucleic acids are
hybridization
probes labeled with fluorescent dyes, such as, for example, oligonucleotides
whose
binding to a nucleic acid target can be detected by the principle of
fluorescence
resonance electron transfer (FRET) (WO 97/46707). This entails a so-called
donor
component, for example fluorescein, being excited with light of a particular
wavelength.
If a suitable acceptor component, such as, for example, certain rhodamine
derivatives,
is in the proximity, then resonance energy transfer to the acceptor component
takes
place, so that the acceptor molecule er~nits light of a particular emission
wavelength.
The hybridization probes can be labeled by standard methods at the 5' end, at
the 3' end or else internally. In a preferred embodiment, the various dyes are
bound to
two different hybridization probes which are able to hybridize in proximity
onto the target
nucleic acid. When in this embodiment both hybridization probes are bound to
the
target DNA, then both components of the FRET system are also in mutual
proximity, so
that fluorescence resonance energy transfer can be measured. This makes
indirect
quantification of the target DNA possible.
The two oligonucleotide probes Ean moreover hybridize onto the same strand of
the target nucleic acid, in which case one dye is preferably located on the 3'-
terminal
nucleotide of the first probe, and the other dye is preferably located on the
5'-terminal
nucleotide of the second probe, so that the distance between the two is only a
small
number of nucleotides, and this number can be between 0 and 30. On use of
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fluorescein in combination with a rhodamine derivative such as, for example,
LC-RED
640 or LC-RED 705 (Roche Molecular Biochemicals) it has emerged that the
distances
are advantageously from 0-15, in particular 1-5, nucleotides and, in many
cases, one
nucleotide. While maintaining the nucleotide distances between the dye
components it
U
is also possible to use probes which are conjugated not terminally but
internally to one
of the dyes. In the case of double-stranded target nucleic acids it is also
possible to
employ probes which bind to different strands of the target, as long as a
particular
nucleotide distance of 0 to 30 nucleotides is maintained between the two dye
components.
Methods of the invention which have accordingly proved to be particularly
advantageous are those in which stxA1 and stxA2 are detected with the aid of
fluorescence resonance energy transfer. The invention likewise relates to
hybridization
probes having a sequence or part-sequence as shown in SEQ ID No. 5-8 and to
methods in which these specific hybridization probes are employed for the
detection of
clinically relevant EHEC infections.
A specific embodiment of the invention is thus also represented by
fluorescence-
labeled probe pairs either as shown in SEQ ID No. 5 and 6 or as shown in SEQ
ID
No. 7 and 8, which are advantageously labeled with, in each case, a FRET donor
component such as, for example, fluorescein and with a FRET acceptor component
such as, for example, CyS, LC-RED 640, LC-RED 705 or another rhodamine
derivative.
Correspondingly labeled oligonucleotide combinations are referred to
hereinafter as
°FRET pairs°.
CA 02393223 2002-03-28
_g_
It has proved particularly advantageous to use such FRET pairs for detecting
amplification products during or after a multiplex amplification reaction. In
a particular
embodiment, one of the two amplification primers can at the same time be
labeled with
one of the two dyes employed, and thus contribute one of the two components of
the
FRET.
The use of suitable FRET pairs for detecting multiplex amplification products
makes parallel, so-called real-time monitoring of PCR reactions possible, it
being
possible to find data for generating the amplification product as a function
of the
number of reaction cycles completed. This usually takes place by the
oligonucleotides
of the FRET pair also hybridizing onto the target nucleic acid because of the
reaction
and temperature conditions during the necessary annealing of the amplification
primers
onto the nucleic acid to be detected, and an appropriately measurable
fluorescence
signal being emitted with suitable excitation. It is thus possible on the
basis of the data
a
obtained to determine quantitatively the amount of target nucleic acid
originally
employed.
In another, preferred embodiment, the multiplex amplification products are
detected after completion of the amplification reaction, in which case, after
hybridization
of the FRET pair onto the target nucleic acid to be detected, the temperature
is
increased continuously in a melting curve analysis. At the same time, the
emitted
fluorescence is measured as a function of the temperature and, in this way, a
melting
temperature at which the FRET pair err~ployed no longer hybridizes onto the
sequence
to be detected is determined. If there are mismatches between the FRET pair
employed
and the amplification product, the melting point is significantly depressed.
It is possible
CA 02393223 2002-03-28
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in this way to identify with one FRET pair different target nucleic acids
whose
sequences differ from one another slightly through one or a few point
mutations.
This principle is employed according to the invention in a multiplex
amplification
reaction for detecting EHEC infections, in which there is use of an internal
standard
which differs from the stxA1 or stxA2 wild-type sequence (GeneBank number
X07865)
only in one or two point mutations. It is thus possible to distinguish
amplified target
nucleic acid and amplified internal standard from one another with the aid of
a melting
curve analysis.
In this case, the standard is preferably employed only in small amounts of
about
100 plasmid copies (1.7 ' 10-~ mol), so that a positive signal relating to
amplification of
the internal standard not only indicates that the PCR has not been inhibited
in any way
in the particular mixture, but also represents a check of the sensitivity of
the reaction.
In this case, the standard is preferably employed only in small amounts of
about 100
plasmid copies (1.7 x 10-~ mol), so that a positive signal relating to
amplification of the
,.
internal standard not only indicates that the PCR has not been inhibited in
any way in
the particular mixture, but also represents a check of the sensitivity of the
reaction.
A further aspect of the method of the invention relates to distinguishing
human-
pathogenic stxA2 and swine-pathogenic stxA2e with the aid of the described
melting
curve analysis.
The use of FRET pairs as shown in SEQ ID No. 5 and 6 or 7 and 8 for
determining melting curves or for distinguishing human-pathogenic stx and
swine-
pathogenic stx is in this connection likewise an aspect of the invention. This
preferably
entails use of a FRET pair as shown in SEQ ID No. 7 and 8.
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The present invention additionally relates to kits which comprise various
reagents for carrying out the methods of the invention. Such kits of the
invention usually
comprise amplification primers for carrying out a multiplex PCR as shown in
SEQ ID
No. 1-4. These kits may preferably also comprise hybridization probes, for
example with
sequences as shown in SEQ ID No. 5-8.
Furthermore, these kits may additionally comprise according to the invention
primers and hybridization probes for amplification of DNA of one or more
additional
EHEC virulence factors such as, for example, EHEC intimin, EHEC hemolysin,
EHEC
catalase, EHEC serine protease and EHEC enterotoxin. Finally, all the kits of
the
invention may additionally comprise reagents which are generally suitable for
carrying
out nucleic acid amplification reactions. These are preferably, but not
exclusively,
special buffers, Taq polymerase and deoxyribonucleotides.
Brief description of the figures:
Figures 1 and 2 show a melting curve analysis as described in example 2. The
first derivative of the measured fluorescence is in each case depicted as a
function of
the respective temperature, measured with a FRET pair composed of fluorescein
and
LC-RED 640 for detecting stxA1 (figure 1 ) and a FRET pair composed of
fluorescein
and LC-RED 705 for detecting stxA2 (figure 2).
Example 1: DNA isolation from bacterial cultures and stool samples
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Bacterial cultures were worked up after overnight culture in TSB broth (casein
peptone, pancreatin digest 17.0 gll; sox meal peptone, papain digest 3.0 g/1;
sodium
chloride 5.0 g11; dipotassium hydrogen phosphate 2.5 g/1; glucose 2.5 gll)
with the aid of
a commercial DNA extraction method (QIAamp DNA Mini Kit, Qiagen, Catalog
No. 51304). For this purpose, 200 ,u1 of the bacterial suspension were
incubated with
20 ,u1 of proteinase K and 200 ,u1 of ATL buffer at 56°C for 10 min.
200 ,u1 of 96%
ethanol were then admixed with the suspension. The solution was then put onto
a
QIAamp spin column and centrifuged at 6 000 g for 1 min. The columns were then
washed once with 500 ~I each of AW1 and AW2 buffers (bench centrifuge 20 000
g).
After the second washing step, the column was centrifuged until dry once. The
purified
DNA was subsequently eluted with 200 ,u1 of AE buffer (10 mM TrisIHCC 0.5 m
MEDT
A pH 9.0). Before use in the PCR reaction, the DNA concentration and purity
were
determined in a photometer (spectrum from 260 nm to 320 nm). A maximum of 500
ng
of template DNA were employed for each PCR mixture. In background
investigations, a
DNA equivalent to 10' bacteria (corresponds to about 55 ng of DNA) was
employed.
PCR investigations were carried out directly on stool samples using a special
stool kit (QIAamp DNA Stool Kit, containing the same buffers as the QIAamp DNA
Mini
Kit). For this purpose, 200 mg of stool X200 ,u1 in the case of diarrheal
stools) were
thoroughly mixed with 600 ,u1 of ASL buffer. In parallel with this, 300 mg of
matrix AX
(adsorbent for inhibitors in stool samples) are resuspended in 900 ,u1 of the
same buffer.
This suspension was then added to the dissolved stool sample and thoroughly
mixed.
Subsequently, the homogenate was incubated at 70°C for 5 min. The
matrix AX and
undissolved stool particles were then pelleted by a centrifugation step at 20
000 g for
CA 02393223 2002-03-28
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3 min. 200 ,u1 of the supernatant were then mixed in analogy to the above-
mentioned
QIAamp DNA mini protocol with 20 ~I of proteinase K and likewise incubated at
56°C
for 10 min. Subsequently, entirely in analogy to the procedure for bacterial
cultures,
using the same buffers, the DNA was bound to a QIAamp spin column, washed,
eluted
and measured in a photometer. The same amounts of DNA as described above were
employed in the subsequent PCR reaction.
Example 2: Amplification and identification of the amplification products
A multiplex PCR for detecting stxA1 and stxA2 in DNA isolated by one of the
methods of example 1 was carried out in the LightCycler system (Roche
Molecular
Biochemicals) in accordance with the rr~anufacturer's information. The
amplification
product was detected according to two different protocols either with the aid
of
SybrGreen as double-stranded DNA-binding agent or, alternatively, with the aid
of
FRET hybridization probes.
All the primers and hybridization probes used were HPLC-purified and were
stored in stock solutions of 100 pMlul (primers) or 3 pMlul (probes). The
primers
employed were selected in this case so that it was possible to amplify a 418
by
fragment of stxA1 (nucleotide position 598-1015, primers as shown in SEQ ID
No. 1
and 2) and a 264 by fragment of stxA2~'(nucleotide position 679-942, primers
as shown
in SEQ ID No. 3 and 4).
The hybridization probes employed for the detection were labeled by standard
protocols. For detecting stxA1, an oligonucleotide as shown in SEQ ID No. 5
was
labeled at the 3' end with fluorescein and an oligonucleotide as shown in SEQ
ID No. 6
CA 02393223 2002-03-28
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was labeled at the 5' end. An oligonucleotide as shown in SEQ ID No. 7 was
labeled at
the 3' end with fluorescein and an oligonucleotide as shown in SEQ ID No. 8
was
labeled at the 5' end with t_C-RED 705 .as hybridization probes for detecting
stxA2.
Detection with SybrGreen:
2.0 ,u1 of DNA master SYBR Green I (Roche Molecular Biochemicals, containing
buffers, Taq DNA polymerase, dNTPs, MgCl2 and SYBR Green I dye)
pM of each primer employed as shown in SEQ ID No. 1-4
2.4 ~cl of 25 mM MgCl2 (working concentration 4 mM)
10 u1 of DNA
,u1 complete mixture
Detection using hybridization probes:
2.0 ,u1 of DNA master for hybridization probes (Roche Molecular Biochemicals,
containing buffers, Taq polymerase, dNTPs and MgClz)
10 pM of each primer employed as shown in SEQ ID No. 1-4
3 pM of each hybridization probe employed as shown in SEQ ID No. 5 and 6 for
stxA1 of SEQ ID No. 7 and 8 stxA2
2.4 ,u1 of 25 mM MgCl2 (final concentration 4 mM)
8.0 u1 of DNA '~
20 ~cl complete mixture
Both mixtures were run with the same PCR program and terminated with
a melting curve:
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Temperature cycles:
95°C 120 sec denaturation at the start of the program
95°C 1 sec
55°C 5 sec (touchdown from 60°C to 55°C in 5 steps of 1
°C)
72°C 20 sec
45 cycles
The melting curves were constructed after previous brief denaturation at
95°C in
an interval from 50°C to 95°C in 0.2°C steps with
continuous fluorescence
measurement on channel 1 (SYBR Green), channel 2 (LC-Red 640) or channel 3 (LC-
Red 705) with the aid of the LightCycler software 3Ø
In SYBR Green mode, the specificity of PCR products from stool samples was
found via the melting point compared with a control of stx-positive bacteria,
in particular
in order to be able to distinguish the amplification product from nonspecific
primer
dimers. In addition, all the results were verified by gel electrophoresis.
In the case of the FRET hybridization probe format it was necessary to employ
the color compensation file of the LightCycler 3.0 software to avoid crosstalk
effects
between channel 2 and 3. The results of a typical experiment are disclosed in
figure 1
and 2:
Figure 1 shows the temperature dependence of the fluorescence from mixtures
with different initial DNA concentrations in channel 2 (LC-RED 640) for
detecting stxA1;
figure 2 shows the fluorescence of the same samples measured in channel 3 (LC-
RED
705) for detecting stxA2. In each case, the first derivative of the
fluorescence measured
as a function of the particular melting curve temperature in accordance with
the
CA 02393223 2002-03-28
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information from the LightCycler manufacturer is depicted. The temperatures of
the
curve maxima found thus correspond to the melting points of the respective
hybridization probes.
Example 3: Sensitivity
DNA of the stx1 and stx2-positive E. coli strain EDL 933 was extracted and
quantified by photometry as in example 1. Based on the assumption that 2 x 108
bacteria contain about 1 ,ug of DNA, the number of bacteria worked up was
inferred and
serial dilutions were set up. Cultured stool samples from routine diagnoses,
which were
free of intestinal pathogens, were processed by the method described above, as
background. It was in this case possible to detect in multiplex mixtures as in
example 2
equivalents of at least about 1.8 stx-positive bacteria in a background of
about 1 x 10'
stx-deficient bacteria in a reaction mixture.
Example 4: Specificity - detection of human- and swine-pathogenic EHEC
48 human isolates of various serotypes, whose genotype was unknown at the
time of the invention but whioh had already been characterized as EHEC strains
by
other, prior art methods, were investigated as in example 2 in the FRET
hybridization
probe mode. In addition, 3 isolates from pigs with E. coli edema disease were
investigated. The result is shown in table 1:
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Table Detection man- and ine-patho~nic stx
1: of hu sw e
S a Code No. Source Serotype stx, stx2 Tm stxA2
r i
a I
No.
1 485/98 CI 0145:H' + - -
2 531 /98 C I O 145: + - -
H'
3 563/98 CI 0113:HNT + - -
4 633/98 CI 026:H' - + 72C
741198 CI 0121:H' - + 72C
6 742/98 CI 08:H' - + 63C
7 768/98 CI 030:H21 - + 72C
8 802198 CI 0157:H' - + 72C
9 1115/98 CI 0157:H' + + 72C
1168198 CI 0128:H' + + 63C
11 1211/98 CI 060:H' - + 63C
12 1244198 CI 06:H8 -
+ 72C
13 1273198 CI 06:H8 -
+ 72C
14 1295/98 CI ONT:H' - + 72C
1306/98 CI 0157:H' + + 72C
16 1568/98 CI 0103:H' + - -
17 1613198 CI ONT:HNT + - -
18 1695198 CI 0103:H' + - -
19 1760/98 CI 0129:H' + + 63C
1762198 CI 0129:H' + + 63C
21 1771/98 CI 0113:H2 + + 63C
22 54/99 C I O 103: + - -
H 18
23 90199 CI O 57:H' - + 72C
24 109199 CI 0157:H' - + 72C
143/99 CI 092:H32 - + 63C
26 144/99 CI 092:H32 - + 63C
27 159/99 CI 076:H' + + 63C
28 197199 CI 0128:HNT + + 63C
29 209/99 CI ONT:HNT - + 72C
240199 CI 030:HNT - + 72C
31 285/99 CI 0145:HNT + - -
32 363199 CI ONT:H' + + 63C
33 497/99 CI 0103:H4 + - -
34 516/99 CI ONT:H' + - -
575199 CI 0157:H' - + 72C
36 576199 CI 0157:H' - + 72C
37 594/99 CI ONT:H' + - -
38 649199 CI ONT:H9 - + 72C
39 680/99 CI 0157:H' + + 72C
707199 CI ONT:HNT + + 63C
41 713/99 CI 0115: + - -
H 10
42 720/99 C I O 111: + - -
H'
CA 02393223 2002-03-28
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S a Code No. Source Serotype stx, stx2 Tm stxA2
r
i
a
I
No.
43 789/99 CI 0103:HNT + - -
44 791/99 CI 091:HNT + - -
45 809/99 CI ONT:HNT + - -
46 826!99 CI ONT:HNT + - -
47 827199 CI ONT:HNT - + 72C
48 834/99 CI ONT:HNT + - -
49 A 3473-1198 ED 0139:H1 - + 63C
50 A 3621-2198 ED 0139:H1 - + 63C
51 82812/99 ED 0139:H1 - + 63C
CI = clinical isolate
Ed = edema disease
It was in this case possible to identify all the human isolates as stx-
positive, with
detection only of stx1 in 18 strains, only of stx2 in 19 strains and of both
genes in 11
strains. The three pig isolates were likewise stx2-positive.
On use of the hybridization samples of the invention as shown in SEQ ID No. 7
and 8, differences in the melting temperatures of stxA2 were measured in
different
isolates: melting temperatures of 71-7~,°C were found for stxA2 in
amplicons of 18 of
the 30 stxA2-containing human isolates. This temperature is identical to the
Tm found
in preceding experiments for cloned stxA2 DNA from human-pathogenic strains.
Melting temperatures of about 63°C were found for the DNA of the
remaining 12 stxA2-
containing human isolates and for the DNA from the three swine-pathogenic
strains,
which certainly contain the stx28 allele. It can be concluded from the
identical Tm that
the 12 human-pathogenic isolates are attributable to swine-pathogenic EHEC
strains
and presumably likewise contain the stx2, allele. This supposition was
confirmed by
sequence analysis of the PCR products from the corresponding 12 isolates.
CA 02393223 2002-03-28
-I8-
Overall, this example shows that both human-pathogenic and swine-pathogenic
EHEC pathogens can be identified with the aid of the method of the invention.
Example 5: Specificity - avoidance of false-positive results
Specificity tests were carried out on 32 stx-negative bacterial strains listed
in
table 1. For this purpose, DNA was extracted as in example 1 from appropriate
,.
overnight cultures. The isolated DNA was subsequently investigated as in
example 2
for the presence of stxA1 and stxA2 using the SybrGreen mode. The result was
always
unambiguously negative. As inhibition control, the DNA was mixed with DNA of
the
stx1- and stsx2-positive E. coli strain EDL 933 in a parallel mixture and
tested for stx1
and stx2 in the same run, unambiguously positive signals being obtained
without
exception.
CA 02393223 2002-03-28
- 19-
Table 2: Bacterial isolates tested
in ~_the_ specifics test
Aeromonas h dro hilia CLINICAL ISOLATE
Bacillus subtilis ATCC 6633
Bacillus subtilis ATCC 6051
Cam lobacter coli CLINICAL ISOLATE
Cam lobacter e'uni ATCC 33560
Candida albicans ATCC 10231 T a 3
Citrobacterfreundii CLINICAL ISOLATE
Enterobacter cloacae CLINICAL ISOLATE
Enterococcus faecalis ATCC 10541
Enterococcus faecalis CLINICAL ISOLATE
Enterococcus faecium ATCC 19434
Enterococcus faecium CLINICAL ISOLATE
Escherichia coli ATCC 25922
EAEC O,-42 CLINICAL ISOLATE
EIEC 460858 CLINICAL ISOLATE
EPEC E 2348169 CLINICAL ISOLATE
ETEC CLINICAL ISOLATE
Helicobacter loci CLINICAL ISOLATE
Klebsiella neumoniae ATCC 10031
Mo anella mo anii CLINICAL ISOLATE
Plesiomonas shi elloides CLINICAL ISOLATE
Proteus mirabilis ATCC 1453
Proteus vu! aris CLINICAL ISOLATE
Pasteurella canis CLINICAL ISOLATE
Pseudomonas aeru inosa ATCC 27853
Salmonella enteritidis CLINICAL ISOLATE
Salmonella t himurium CLINICAL ISOLATE
Shi ella flexneri CLINICAL ISOLATE
Sta h lococcus aureus CLINICAL ISOLATE
Sta h lococcus a idermidis ATCC 12228
Stre tococcus a alactiae CLINICAL ISOLATE
Yersinia enterocolitica CLINICAL ISOLATE
This example thus shows that the method of the invention is suitable for
specific detection of EHEC infections.
CA 02393223 2002-03-28
-20-
SEQUENCE LISTING
<110> CYTONET GMBH & CO. K~
<120> Multiplex PCR for detecting EHEC infections
<130> 529800da
<140>
<141>
<160> 8
<170> Patentln Ver 2.1
<210> 1
<211> 24
<212> DNA
<213> Escherichia coli
V
<400> 1
agtogtacgg
ggatgcagat
aaat
24
<210> 2
<211> 24
<212> DNA
<213> Escherichia coli
<400> 2
ccggacacat agaaggaaac tcat 24
<210> 3
<211> 20
<212> DNA
<213> Escherichia coli
<400> 3
ttccggaatg caaatcagtc 20
<210> 4
<211> 21
<212> DNA
<213> Escherichia coli
<400> 4
cgatactccg gaagcacatt g 21
CA 02393223 2002-03-28
-21-
<210> 5
<211> 30
<212> DNA
<213> Escherichia coli
<400> 5
ctgtcacagt aacaaaccgt aacatcgctc 30
<210> 6
<211> 24
<212> DNA
<213> Escherichia coli
<400> 6
tgccacagac tgcgtcagtg aggt 24
<210> 7
<211> 28
<212> DNA
<213> Escherichia
coli
<400> 7
agagcagttc tgcgttttgt cactgtca 28
<210> 8
<211> 23
<212> DNA
<213> Escherichia coli
<400> 8
agcagaagcc ttacgcttca ggc 23
